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IRAP, REMAP and ISSR Fingerprinting in Newly Formed Hexaploid Tritordeum (X Tritordeum Ascherson et Graebner) and Respective Parental Species

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Retrotransposon (RTN)-based markers, such as the inter-retrotransposon amplified polymorphism (IRAP) and the retrotransposon-microsatellite amplified polymorphism (REMAP), are highly informative, multilocus, and reveal insertion polymorphisms among individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, and detection of genetic rearrangements induced by allopolyploidization. The hexaploid tritordeum (HchHchAABB; 2n = 6x = 42) is an allopolyploid produced from crosses between wild barley (Hordeum chilense Roem. et Schultz.) (HchHch; 2n = 2x = 14) and durum wheat (Triticum turgidum L. conv. durum) (AABB; 2n = 4x = 28). With this study, we carried out the DNA fingerprinting of two newly formed hexaploid tritordeum lines (HT22 and HT27) and their respective parents, line H1 of H. chilense and line T81 of durum wheat, based on IRAPs, REMAPs and inter-simple sequence repeats (ISSRs), in order to detect potential rearrangements in tritordeum derived from polyploidization. The amphiploid nature of the HT22 and HT27 individuals was successfully confirmed after fluorescence in situ hybridization (FISH), which was performed on their mitotic chromosome spreads with genomic DNA from H. chilense and 45S ribosomal DNA (rDNA), simultaneously, as probes. Six combinations of LTR (long terminal repeat) primers and seven combinations of one LTR and one SSR (simple sequence repeat) primers successfully produced IRAPs and REMAPs, respectively, in both tritordeum lines, and their respective parents. ISSRs were produced with three SSR primers (8081, 8082, and 8564). The analysis of the presence/absence of bands among the tritordeum lines and the respective parents allowed the detection of polymorphic bands: (1) shared by tritordeum and one of the parents; (2) exclusively amplified in tritordeum; and (3) exclusively present in one of the parents. Once no polymorphism was detected among the individuals of each parental species, the polymorphic bands that fit into the second and third cases probably constituted rearrangements in the newly formed tritordeums that arose in response to allopolyploidization, which resulted from the loss of parental bands or, conversely, from the appearance of novel bands not seen in the parental species. Most of the polymorphic IRAPs in tritordeum were shared with the female parent (H. chilense), while most of the polymorphic REMAPs and ISSRs were common to the male parent (durum wheat), but globally, most of the bands inherited by tritordeum had a wheat origin. In conclusion, these dominant markers were successful for DNA fingerprinting and detection of rearrangements in newly formed tritordeum derived from responses to allopolyploidization.

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This work was partially supported by the HY-WHEAT project - International Consortium (P-KBBE/AGR-GPL/0002/2010), funded by the COMPETE program, QREN, EU, and the Portuguese Foundation for Science and the Technology (FCT).

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Correspondence to José Lima-Brito.

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Cabo, S., Carvalho, A., Rocha, L. et al. IRAP, REMAP and ISSR Fingerprinting in Newly Formed Hexaploid Tritordeum (X Tritordeum Ascherson et Graebner) and Respective Parental Species. Plant Mol Biol Rep 32, 761–770 (2014).

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